| Description | Aladdin's Anti-V5 Affinity Gel, also known as Anti-V5 IP Gel, Anti-V5 immunoprecipitation gel or Anti-V5 agarose gel, is agarose gel covalently coupled with high-quality mouse monoclonal antibody that recognizes the V5 tag sequence (GKPIPNPLLGLDST). This product can specifically bind V5-tagged Aladdin's Anti-V5 Affinity Gel, also known as Anti-V5 IP Gel, Anti-V5 immunoprecipitation gel or Anti-V5 agarose gel, is agarose gel covalently coupled with high-quality mouse monoclonal antibody that recognizes the V5 tag sequence (GKPIPNPLLGLDST). This product can specifically bind V5-tagged proteins expressed in animals, plants, and microorganisms, and can be used for immunoprecipitation (IP) and purification of V5-tag fusion proteins or their protein complexes.V5-tag is a peptide consisting of 14 amino acids (GKPIPNPLLGLDST) isolated from monkey parainfluenza virus V5, which can be fused either to the N-terminus or the C-terminus of a target protein to facilitate protein purification and immunoassays. V5-tag usually does not interact with the target protein, and in most cases does not affect the function of the target protein; With Aladdin's V5 antibodies, Anti-V5 magnetic beads, or Anti-V5 affinity gel, V5-tagged proteins can be used for examination of protein expression level and subcellular localization, protein purification, and immunoassays.This product has high binding capacity and strong specificityPrecautions：This product must be fully resuspended by inverting the tube several times prior to use.This product contains a small amount of preservative, which does not affect routine IP assays and protein purification. But for special experiments that might be interfered by the preservative, the gel should be washed 3 times with appropriate solutions such as TBS prior to use.For immunoprecipitation assays, it is recommended to include both positive and negative controls. Protein samples should be purified as soon as possible after collection and should always be placed at 4℃ or on ice to minimize protein degradation or denaturation. Protein degradation can be inhibited by adding appropriate protease inhibitors, such as Protease Inhibitor Cocktail for General Use, Protease and Phosphatase Inhibitor Cocktail for General Use (MS-safe, 50X), Protease Inhibitor Cocktail for Mammalian Cell and Tissue Extracts, Protease and Phosphatase Inhibitor Cocktail for Mammalian Cell and Tissue Extracts, etc.High concentrations of DTT, mercaptoethanol, guanidine hydrochloride, etc. may have a certain effect on the binding of this product to V5-tagged proteins, but Cell lysis buffer for Western and IP, RIPA Lysis Buffer or NP-40 Lysis Buffer are applicable to this product. For the main features and differences of various lysis buffers produced by and the selection of lysis buffers, please refer to the website at http://www.aladdin-e.com/support/lysis-buffer.htm.If the insoluble substance in protein samples can not be removed completely by centrifugation, samples can be filtered with a 0.45µm filter before performing the assay.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1.Preparation of protein samplesa.Lysis cells or tissues with appropriate lysis buffer, such as ’s Cell Lysis Buffer for Western and IP. Under certain circumstances, ’s RIPA Strong Lysis Buffer, RIPA Medium Lysis Buffer, or RIPA Weak Lysis Buffer can also be used. Other lysis buffers with a pH6-8 can also be used.b.After lysis and centrifuge, keep the supernatant at 4℃ or on ice for subsequent use. We recommend performing the subsequent procedures on the same day. Otherwise, make aliquots and store them at -80℃ for future use.2.Preparation of Anti-V5 Affinity GelAs the Anti-V5 Affinity Gel is stored in a protective solution containing 50% glycerol, it needs to be washed properly before adding protein samples.a.Gently resuspend the Anti-V5 Affinity Gel to be homogeneous. Transfer 20µl of gel suspension per 100µl of protein sample into a clean centrifuge tube. Note: It is easier to aspirate the gel suspension using a large aperture pipette tip (e.g., by cutting off part of the tip with scissors).b.Add 1X TBS to a final volume of 0.5ml, and gently resuspend the Anti-V5 Affinity Gel by pipetting or vortex. Centrifuge at 6000×g for 30 seconds at 4℃, remove the supernatant carefully without disturbing the gel. Repeat this step twice.c.Resuspend the Anti-V5 Affinity Gel with 1X TBS equal to the initial volume of gel suspension (e.g., if 20µl of gel suspension is taken, add 20µl of 1X TBS).3.Protein binding1)Add 20µl of washed Anti-V5 Affinity Gel per 100µl of protein sample, mix well, and incubate at 4℃ for 1-2 hours or overnight with gentle shaking on a rotary mixer.2)After incubation, centrifuge at 6000×g for 30sec at 4℃ and remove the supernatant carefully without disturbing the gel. Note: A portion of supernatant can be reserved in a clean microfuge tube for examination of the binding results.3)Gently resuspend the Anti-V5 Affinity Gel in 500µl of 1X TBS and place in an ice bath on a shaker for 5 minutes. Centrifuge at 6000×g for 30 seconds at 4℃ and carefully remove the supernatant. Repeat the wash thrice, then keep the sample on ice for elution. 4.ElutionBased on the features of the target protein or downstream applications, one of the following three elution methods can be used.1)Competitive elution with the V5 peptide. This is a non-denaturing elution method with a high elution efficiency, and the eluted proteins do not contain the light and heavy chains of V5 antibody.a.Preparation of V5 peptide elution buffer: Dissolve the V5 Peptide (, P9813) in 1X TBS buffer to a final concentration of 150µg/ml, or dilute the 5mg/ml V5 peptide solution with 1X TBS buffer to a final concentration of 150µg/ml. b.For every 20µl of initial gel volume, add 100µl of V5 Peptide elution buffer (150µg/ml). Resuspend the gel gently and incubate for 30-60 minutes at room temperature on a rotary mixer, or 1-2 hours at 4℃. To improve the elution efficiency, increase the incubation time or repeat the elution. c.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and transfer the supernatant to a new microfuge tube. The supernatant contains the V5-tagged protein and its protein complex.d.Store the supernatant at 4℃ for immediate use, or -20℃/ -80℃ for long-term storage. 2)Acidic elution: This method is non-denaturing, relatively fast and efficient. The eluted proteins also retain their original biological activity, which facilitates subsequent analysis and detection.a.Preparation of acidic elution buffer (0.1M Glycine-HCl, pH3.0) and neutralization buffer (0.5M Tris-HCl, pH7.4, 1.5M NaCl). b.For every 20µl of initial gel volume, add 100µl of acidic elution buffer. Resuspend the agarose gently and incubate for 5 minutes at room temperature on a rotary mixer.Note: The incubation time should not exceed 15 minutes.c.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and transfer the supernatant to a new microfuge tube. Immediately add 10µl of neutralization buffer and mix well. d.To achieve the maximum elution efficiency, repeat steps b-c and combine the supernatant containing the V5-tagged protein and its protein complex. e.Store the supernatant at 4℃ for or immediate use, or -20℃/-80℃ for long-term storage. Note 1: The elution efficiency of the acidic elution method might be lower than the other two elution methods.Note 2: The elution efficiency of the acidic elution method depends on characteristics of the target protein. To obtain a higher elution efficiency, the pH of the acidic elution buffer can be optimized from 2.5 to 3.1. The pH or amount of neutralization buffer needed to neutralize the eluates should also be adjusted appropriately.3)Elution with the 1X SDS-PAGE loading buffer: This method is a denaturation method, and the obtained proteins are suitable for SDS-PAGE electrophoresis or WB blot analysis.a.Preparation of SDS-PAGE loading buffer: We recommend using ’s SDS-PAGE Sample Loading Buffer (2X). Usually, the SDS-PAGE protein loading buffer contains a reducing agent such as DTT, and the eluted protein sample will contain the light chain and heavy chain of the V5 antibody.b.For every 20µl of initial gel volume, add 20µl of 2X SDS-PAGE sample loading buffer, mix well and heat at 95℃ for 5 minutes. c.Centrifuge at 6000×g or 4℃ for 30 seconds. Take the supernatant for SDS-PAGE electrophoresis or Western blot analysisNote: The Agarose cannot be reused because SDS in the loading buffer destroys V5 antibodies.FAQ: Problem Possible Causes Solution Large amount of tagged protein found in the flow through. Binding time is not enough. If using batch method, increase the binding time experimentally; If using column method, use a lower flow rate when loading samples. Column is overloaded. Reduce the amount of the sample added to the gel or increase the amount of gel. Tag is not accessible to gel. Expose the epitope tag by adding low amount of denaturant to the protein extract (dialysis may be needed before applying onto gel), or fuse thetag to the other terminus of the target protein. Gel has not been regenerated since last purification. Perform gel regeneration procedure prior to binding. Reagent compatibility problem. Dialyze the sample against TBS before purification procedure. The target protein has been degraded. 1.Prepare fresh lysates. Avoid using frozen lysates.2.Perform purification at lower temperature, such as 4℃.3.Use appropriate protease inhibitors in the lysate or increase their concentrations to prevent degradation of the fusion protein. Very few or notagged protein exists in the eluate. Protein is not completely eluted. Change elution methods. No target protein expressed. Make sure the protein of interest contains the tag by Western blot or dot blot analyses. Very low protein expression level. 1.Use larger volume of cell lysate.2.Optimize expression conditions to raise the protein expression level. Washes are too stringent. 1.Reduce the number of washes.2.Avoid adding high concentrations of NaCl to the mixture.3.Use solutions that contain less or no detergent Incubation times are inadequate. Increase the incubation times with the affinity gel (from several hours to overnight). Interfering substance is present in sample. 1.Lysates containing high concentrations of DTT, 2-mercaptoethanol, or other reducing agents may destroy antibody function, and must be avoided.2.Excessive detergent concentrations may interfere with the antibody-antigen interaction. Detergent levels in buffers may be reduced by dilution. Detection system is inadequate. If Western blot detection is used:1.Check primary and secondary antibodies using proper controls to confirm binding and reactivity.2.Verify that the transfer was adequate by staining the membrane with Ponceau S.3.Use fresh detection substrate or try a different detection system. Multiple protein bands found in the eluate. The protein is not stable at room temperature. Purify the target protein at lower temperature, such as 4℃. Protein degradation due to proteases activity during purification process. Add protease inhibitors to cell lysate. Non-specific binding. 1.Prepare cell lysate again.2.Add additional wash steps. Background is too high. Proteins bind nonspecifically to the monoclonal antibody, the gel beads, or the microcentrifuge tubes. 1.Pre-clear lysate with Mouse IgG-Agarose to remove nonspecific binding proteins.2.After suspending beads for the final wash, transfer entire sample to a clean microcentrifuge tube before centrifugation. Washes are insufficient. 1.Increase the number of washes.2.Prolong duration of the washes, incubating each wash for at least 15 minutes.3.Increase the salt and/or detergent concentrations in the wash solutions.4.Centrifuge at lower speed to avoid nonspecific trapping of denatured proteins from the lysate during the initial centrifugation of the affinity gel complexes... Read More | Inquire | Product contentF665766Component5 mLStorageF665766A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665766BddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Product contentF665766Component5 mLStorageF665766A2×Fast Probe Mixture5×1 mL-20℃. Avoid freeze/thaw cycle.F665766BddH2O5×1 mL-20℃. Avoid freeze/thaw cycle.Product IntroductionFast Probe Mixture is a pre-mixed system for real-time fluorescence PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, including Fast Taq DNA Polymerase, PCR Buffer, dNTPs, Mg2+ and so on, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequence and cDNA target sequence after RNA reverse transcription. The Fast Taq DNA Polymerase contained in this product can effectively reduce the non-specific amplification generated by the non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme only needs to be incubated at 95 ℃ for 30 s. The whole PCR reaction process can save about 40 minutes compared with the ordinary reaction, which greatly shortens the reaction time of PCR. The combination of unique PCR buffer system and fast hot start enzyme effectively inhibits the generation of non-specific products and significantly improves the PCR amplification efficiency with stronger fluorescence signal, higher sensitivity and wider linear range. The product has a wide range of applications and can be used for both normal and rapid quantitative PCR programs.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration (F665766):Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments that require Low ROX calibration (F665768):ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments that require High ROX calibration (F665774):ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.2. Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.UsageThe following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction systemReagents50 µl Reaction systemFinal concentration2×Fast Probe Mixture25 µl1×Forward Primer，10 µM1 µl0.2 µM¹⁾Reverse Primer，10 µM1 µl0.2 µM¹⁾Probe，10 µM1 µl0.2 µM²⁾Template DNA2 µl³⁾ 50×Low ROX or High ROX（optional）⁴⁾1 µl1×ddH₂Oup to 50 µlNote: 1) Usually the primer concentration of 0.2µM can get better results, and 0.1-1.0µM can be used as a reference for setting the range. 2) The final concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the instruction manual of the instrument or the specific requirements of the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.2. PCR reaction program:A two-step PCR reaction program is recommended, and this program is set up using the ABI 7500 Fluorescent Quantitative PCR Instrument as a reference.Note: 1) The enzyme used in this product must be pre-denatured at 95°C for 30s to achieve enzyme activation. Under this condition, most of the templates can be well unchained. For templates with high GC content and complex secondary structure, the pre-denaturation time can be extended to 1-4 minutes in order to make the starting template fully unchained.(2) It is recommended to use two-step PCR reaction program, if you can not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out a three-step PCR amplification, annealing temperature, please use the range of 56 ℃ - 64 ℃ for as a reference for the setting... Read More | Product contentG665787Component5 mLStorageG665787A2×GoldStar Probe Mixture (UNG)5×1 mL-20℃. Avoid freeze/thaw cycle.G665787B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.G665787CddH2O 5×1 mL -20℃. Avoid freeze/thaw cycle. Product Introduction2× Product contentG665787Component5 mLStorageG665787A2×GoldStar Probe Mixture (UNG)5×1 mL-20℃. Avoid freeze/thaw cycle.G665787B50×High ROX200 µL-20℃. Avoid freeze/thaw cycle.G665787CddH2O 5×1 mL -20℃. Avoid freeze/thaw cycle. Product Introduction2× GoldStar Probe Mixture (UNG) is a premixed system dedicated to real-time fluorescence quantitative PCR by probe method (TaqMan, Molecular Beacon, etc.), with a concentration of 2×, containing GoldStar Taq DNA polymerase, PCR Buffer, dNTPs (dTTP is all replaced by dUTP), UNG enzyme and Mg2+, which is easy and convenient to operate. It is mainly used for the detection of genomic DNA target sequences and cDNA target sequences after RNA reverse transcription, such as gene expression analysis, copy number analysis and SNP genotype analysis. This product utilizes the dUTP-UNG anti-pollution system, which adds dUTP during the preparation of the PCR reaction system, thus forming an amplification product containing dU bases. This product can be eliminated by the UNG enzyme in the PCR system before the next PCR reaction. This effectively removes residual contamination of the PCR product and greatly reduces false positives due to contamination of the amplification product.UNG enzyme can be inactivated at the pre-denaturation step in the PCR cycle, and therefore will not affect the formation of new PCR products containing dU bases. The GoldStar Taq DNA Polymerase contained in this product is a chemically modified, new high-efficiency hot-start enzyme, which has no polymerase activity at room temperature, effectively avoiding non-specific amplification due to non-specific binding of primers and templates or primer dimerization at room temperature, and the activation of the enzyme must be incubated at 95°C for 10 minutes. The unique combination of PCR buffer system and hot-start enzyme significantly improves the amplification efficiency of PCR with stronger fluorescent signal and higher sensitivity to detect single-copy templates. A wider linear range and more accurate quantification of the target gene can be obtained by using this product.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration (G670150):Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others.Instruments requiring Low ROX calibration(G665780):ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 system. Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and more.Instruments requiring High ROX calibration(G665787):ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attentionBefore use, please mix gently by turning up and down, avoid foaming as much as possible, and use after brief centrifugation.Avoid repeated freezing and thawing of this product, repeated freezing and thawing may degrade the product performance. This product can be stored for long term at -20℃, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.UsageThe following examples are conventional PCR reaction systems and reaction conditions, which should be improved and optimized according to the template, primer structure and target fragment size in actual operation.1.PCR reaction systemReagents50 µl Reaction systemfinal concentration2×GoldStar Probe Mixture（UNG）25 µl1×Forward Primer，10 µM1 µl0.2 µM¹⁾Reverse Primer，10 µM1 µl0.2 µM¹⁾Probe，10 µM1 µl0.2 µM²⁾Template DNA2 µl³⁾ 50×Low ROX or High ROX（optional）⁴⁾1 µl1×ddH₂Oup to 50 µlNote: 1) Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.(2) The concentration of the probe used is related to the fluorescence quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, please refer to the instrument manual or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration in actual use.(3) Usually the amount of DNA template is 10-100ng genomic DNA or 1-10ng cDNA as a reference. Since the templates of different species contain different copy numbers of target genes, the templates can be subjected to gradient dilution to determine the optimal amount of template to be used.(4) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.2.PCR reaction programCaution! The pre-denaturation reaction of this product must be completed at 95°C for 10 minutes!Two-step PCR:Note: 1) The hot-start enzyme used in this product must be activated under the condition of pre-denaturation 95℃, 10min. 2) It is recommended to use two-step PCR reaction program, if you can't get good experimental results due to the use of primers with lower Tm value, etc., you can try to carry out three-step PCR amplification.Three-step PCR:... Read More | Inquire |